PROJECT SUMMARY Neuroblastoma (NB) is an early childhood tumor of the developing sympathetic nervous system that contributes to approximately 15% of childhood cancer-related deaths. Survival rates for the high-risk form of the disease remain at under 50%, emphasizing the critical need for more effective therapies. T-cell based immunotherapies have shown promising results in hematopoietic malignancies, but in solid tumors like NB, the nutrient-poor, immunosuppressive tumor microenvironment (TME) limits the effector function of these lymphocytes. Thus it is imperative to understand how lymphocytes adapt to the TME in order to harness their anti-tumor activity for developing more effective immunotherapies. Using an autochthonous, immunocompetent murine model of high-risk NB (TH-MYCN+/+), our lab identified a significant population of intratumoral invariant natural killer T (iNKT) cells relative to conventional T cells (TCONV), suggesting that iNKT cells may harbor unique features enabling survival in the TME. iNKT cells possess phenotypic and functional features of both innate natural killer cells and memory T cells and engage a number of anti-tumor immune pathways. However, their function in the TME is largely unknown. In TCONV, metabolism and effector function are tightly linked. Upon stimulation, nave and memory T cells shift from oxidative metabolism to rely on glycolysis as they differentiate into effector T cells. However, in the TME, TCONV must compete for limited glucose supplies, placing bioenergetic constraints that limit their anti-tumor function. Indeed, oxidative metabolism enables long-term survival and persistence of TCONV. In contrast, the metabolic properties of iNKT cells are not known, and may provide critical insight into their anti-tumor functions in the TME. Our preliminary analysis suggests that in vitro stimulated iNKT cells uptake less glucose than TCONV, suggesting that iNKT cells may have unique metabolic properties that confer enhanced effector function in the TME. I hypothesize that iNKT cells are uniquely capable of adapting to the TME by relying predominantly on oxidative metabolism, enabling sustained anti- tumor effector function. In Aim 1, I will define the metabolic profiles (1a) and functional properties (1b) of iNKT cells stimulated in TME-like conditions in vitro to assess the individual contributions of glucose and oxygen deprivation on their metabolic and anti-tumor activity relative to TCONV. In Aim 2, I will use a conjugate molecule that fuses an iNKT cell activating ligand to an NB-specific antigen to specifically activate iNKT cells in the murine NB TME and assess the frequency, proliferation, metabolism, and functional features of these iNKT cells (2a) and determine whether this activation of intratumoral iNKT cells controls NB growth and prolongs survival of TH-MYCN+/+ mice. Collectively, the proposed studies will define novel cellular properties of iNKT cells and determine contexts in which iNKT cells may be clinically superior to TCONV, which will have important therapeutic value for the design of more effective immunotherapies for NB and additional solid tumors.